The transcriptome provides the database from which a cell assembles its collection of proteins. Translation of individual mRNA species into their encoded proteins is regulated, producing discrepancies between mRNA and protein levels. Using a new modeling approach to data analysis, a striking diversity is revealed in association of the transcriptome with the translational machinery. Each mRNA has its own pattern of ribosome loading, a circumstance that provides an extraordinary dynamic range of regulation, above and beyond actual transcript levels. Using this approach together with quantitative proteomics, we explored the immediate changes in gene expression in response to activation of a mitogen-activated protein kinase pathway in yeast by mating pheromone. Interestingly, in 26% of those transcripts where the predicted protein synthesis rate changed by at least 3-fold, more than half of these changes resulted from altered translational efficiencies. These observations underscore that analysis of transcript level, albeit extremely important, is insufficient by itself to describe completely the phenotypes of cells under different conditions. Molecular & Cellular Proteomics 3:478 -489, 2004.

The chimeric anti-CD30 IgG1, cAC10, conjugated to eight equivalents of monomethyl auristatin E (MMAE) was previously shown to have potent antitumor activity against CD30-expressing tumors xenografts in mice. Moreover, the therapeutic index was increased by lowering the stoichiometry from 8 drugs/antibody down to 2 or 4. Limitations of such 'partially-loaded' conjugates are low yield (10-30%) as they are purified from mixtures with variable stoichiometry (0-8 drugs/antibody), and heterogeneity as the 2 or 4 drugs are distributed over eight possible cysteine conjugation sites. Here, the solvent-accessible cysteines that form the interchain disulfide bonds in cAC10 were replaced with serine, to reduce the eight potential conjugation sites down to 4 or 2. These Cys-->Ser antibody variants were conjugated to MMAE in near quantitative yield (89-96%) with defined stoichiometries (2 or 4 drugs/antibody) and sites of drug attachment. The engineered antibody-drug conjugates have comparable antigen-binding affinities and in vitro cytotoxic activities with corresponding purified parental antibody-drug conjugates. Additionally, the engineered and parental antibody-drug conjugates have similar in vivo properties including antitumor activity, pharmacokinetics and maximum tolerated dose. Our strategy for generating antibody-drug conjugates with defined sites and stoichiometries of drug loading is potentially broadly applicable to other antibodies as it involves engineering of constant domains.

High bone mass diseases are caused both by activating mutations in the Wnt pathway and by loss of SOST, a bone morphogenetic protein (BMP) antagonist, leading to the activation of BMP signaling. Given the phenotypic similarity between mutations that activate these signaling pathways, it seems likely that BMPs and Wnts operate in parallel or represent components of the same pathway, modulating osteoblast differentiation. In this study, we show that in C3H10T1/2 cells, Wnt-3A and BMP-6 proteins were inducers of osteoblast differ- Sclerostin, the protein product of the sost gene associated with the high bone mass sclerosteosis phenotype, was predicted to be a secreted glycoprotein with homology to the DAN family of bone morphogenetic protein (BMP) 1 antagonists and more distantly to the BMP antagonist noggin (1, 4). We have shown that sclerostin behaved as a BMP antagonist and blocked BMPinduced osteoblastic activity such as alkaline phosphatase (ALP) activity in human and rodent bone cell models (3). In a broader context, BMPs and BMP antagonists have described skeletal roles specifically in chondrocyte differentiation, joint formation, and osteogenesis (5-7). Recently, Fischer et al. (8) and Bain et al. (9) reported that BMP-2 mediated osteoblast differentiation was modulated by a member of the Wnt protein family (8, 9). A novel role for Wnts was implicated in osteogenesis and BMP signaling.Wnt proteins are cysteine-rich, secreted glycoproteins that have been implicated in embryonic development and cellular differentiation, in particular limb patterning and chondrogenesis (10, 11). Wnt proteins activate downstream signaling pathways in target cells through interactions with Frizzled and low density lipoprotein receptors (LRP) co-receptors. The identification of the high bone mass gene, an activating mutation in LRP5, revealed the role of the Wnt pathway in bone formation (2,12).Given the interaction between the BMP and Wnts in osteoblastic cells described in the literature (8, 9), we were interested in investigating whether BMP antagonists, in particular sclerostin, could modify Wnt activity. Our findings show that the sclerostin antagonism of Wnt-3A-induced activity was not due to a direct interaction between the proteins or between sclerostin and the Wnt signaling pathway. We conclude that sclerostin inhibition of Wnt activity is mediated by BMP proteins and that Wnt induction of ALP in C3H10T1/2 cells is dependent on the expression of BMPs. EXPERIMENTAL PROCEDURES Effects

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